A complex-trapping-blocking (CTB) ELISA, using monoclonal antibodies and detecting specifically antibodies directed against foot-and-mouth disease types A, O and C. I. Method and characteristics

A complex-trapping-blocking (CTB) enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies directed against foot-and-mouth disease virus (FMDV) strains A10 Holland, O1 BFS, and C1 Detmold. For each strain two monoclonal antibodies directed against different antigenic sites of FMDV were used. The assay used either infectious, not inactivated antigen or inactivated antigen. We concluded that the CTB-ELISA was sensitive, type-specific, and more reproducible (P less than 0.05) than the serum neutralisation test (SNT). In addition, the test was easy to perform and results could be recorded within 3 hours. The cross-reactivity of bovine reference sera raised against the three FMDV strains was comparable in the CTB-ELISA and the SNT.     

Endurance of immunity against foot-and-mouth disease in cattle after three consecutive annual vaccinations

Protection against foot-and-mouth disease (FMD) and ability to transmit FMD virus to susceptible contact animals were studied in cattle vaccinated three times in annual field campaigns with the Dutch trivalent vaccine. Eighty vaccinated cattle and 16 susceptible controls were intranasally exposed to an aerosol containing a homologous FMD challenge virus (O1 BFS, A10 Holland or C1 Detmold) or a heterologous virus (A5 Modena or C1 Modena). The day after exposure, vaccinated cattle were stabled individually with an FMD-susceptible contact. All cattle challenged with an homologous virus strain at one year (20 head), at two years (10 head) and at three years (30 head) after the last vaccination were protected against the development of clinical signs of disease; one, zero and five cattle of these groups, respectively, transmitted virus to their contacts. In each group, approximately two out of three exposed cattle had virus-positive oropharyngeal fluid samples and seroconverted. The amount of virus recovered from probang samples increased with the time since the last vaccination. Mean antibody titres of cattle that had not been vaccinated for three consecutive years did not change significantly over the last two-year period. All 10 cattle challenged with the vaccine strain-related C1 Modena virus were protected against clinical disease, whereas three out of 10 challenged with the heterologous A5 Modena strain virus one year after the last vaccination contracted FMD and transmitted the virus. Five others (four in the C1 group and one in the A5 group) spread the virus to their contacts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessment using ELISA of the herd immunity levels induced in cattle by foot-and-mouth disease oil vaccines

The development of a liquid-phase blocking sandwich ELISA (LPBE) to measure antibodies (Ab) produced in cattle with the O, A and C foot-and-mouth disease virus (FMDV) types of commercial vaccines used in Argentina is described. The test was specific: 99% of naïve cattle sera (n = 130) gave titres below log₁₀ = 1.2, and none had a titre above log₁₀ = 1.5. Comparative studies with serum neutralization test (SNT) using sera from cattle which received one or more vaccine doses is reported. The overall rank correlation coefficient (Spearman's , r_s) between SNT and LPBE were highly significant (r_s > 0.67, P < 0.0001) for all vaccine strains. LBPE Ab titres on sera collected 90 days post vaccination were compared with results of cattle protection tests by applying a logistic regression. The minimum Ab titres at which 85% and 75% of the cattle were protected for each FMDV type were determined in order to interpret field Ab data in terms of protection. Application of this method allows large scale serological examinations to monitor antibody levels in vaccinated animals as an indirect indicator of the FMD control program status in the field. Its use in the evaluation of commercial batches of FMD vaccine is discussed.     

A serological survey for antibodies against foot-and-mouth disease virus (FMDV) in domestic pigs during outbreaks in Kenya

Foot-and-mouth disease (FMD) is endemic in Kenya and has been well studied in cattle, but not in pigs, yet the role of pigs is recognised in FMD-free areas. This study investigated the presence of antibodies against FMD virus (FMDV) in pigs sampled during a countrywide random survey for FMD in cattle coinciding with SAT 1 FMDV outbreaks in cattle. A total of 191 serum samples were collected from clinically healthy pigs in 17 districts. Forty-two of the 191 sera were from pigs vaccinated against serotypes O/A/SAT 2 FMDV. Antibodies against FMDV non-structural proteins were found in sera from 30 vaccinated and 71 non-vaccinated pigs, altogether 101/191 sera (53 %), and 91 % of these (92/101) also had antibodies measurable by serotype-specific ELISAs, predominantly directed against SAT 1 with titres of 10–320. However, only five high titres against SAT 1 in vaccinated pigs were confirmed by virus neutralisation test (VNT). Due to high degree of agreement between the two ELISAs, it was concluded that positive pigs had been infected with FMDV. Implications of these results for the role of pigs in the epidemiology of FMD in Kenya are discussed, and in-depth studies are recommended.     

The efficacy of FMD vaccine reduced non-structural proteins with a mAb against 3B protein

A monoclonal antibody, 3BIgG, against the prokaryotically expressed foot-and-mouth disease virus (FMDV) non-structural protein (NSP) 3B was obtained. The 3BIgG-sepharose conjugant (3BmAb-6BFF) was prepared by adding the purified 3BIgG into epoxy-activated sepharose 6BFF, incubating with the inactivated FMDV, and then removing the sepharose by centrifugation. The vaccine was made from the supernatant emulsified with oil-adjuvant ISA206. Ten guinea pigs, 26 pigs and six cattle were vaccinated, and a vaccination control group was included without treatment with 3BmAb-6BFF. After 28 days, 9/10 pigs challenged with FMDV were protected, this result was the same as the control group, indicating that the vaccine potency was not reduced after treatment with 3BmAb-6BFF. The other animals were vaccinated weekly for nine weeks, and serum samples were collected to detect 3ABC-antibody titers. The results showed that 3ABC-antibody production was delayed and the positive antibody rates were lower when vaccination was carried out using vaccines treated with 3BmAb-6BFF compared with untreated vaccines. The findings of this study suggest that it is possible to reduce NSPs using a mAb-sepharose conjugant in FMD vaccines without reducing their efficacy.     

A Comparison of Methods for Measuring the Antibody Response in Mice and Cattle following Vaccination against Food and Mouth Disease

We present a comparison of methods for evaluating the potency of foot and mouth disease vaccine in the laboratory. The anti-FMDV antibodies (Ab) in vaccinated mice were tested by liquid phase (lp) ELISA, solid phase (sp) ELISA and virus neutralization (VN), and were compared with the Ab titres detected by lpELISA, which is the official test in Argentina for testing the potency of FMD vaccines and protection against a virulent challenge in cattle. The results demonstrated that it is possible to relate the Ab levels induced in vaccinated mice with both the Ab and protective responses elicited in cattle. Furthermore, it was found that the anti-FMDV Ab titres in mice detected by lpELISA 14 days after vaccination should be an accurate parameter for predicting the results of the challenge test in cattle. Thus, this test in mice appears to be an inexpensive and rapid alternative for testing FMD vaccines in cattle.     

豚鼠动物模型评价牛口蹄疫Asia-1型灭活疫苗效力的研究

以豚鼠为试验动物模型,探索一种应用豚鼠替代牛进行牛口蹄疫Asia-1型灭活疫苗效力检验的方法.豚鼠和牛同步对6批牛口蹄疫Asia-1型灭活疫苗进行PD50效力检验,其中2批进行重复性试验.豚鼠分别在免疫后7、14、21和28天采血检测Asia-1型的中和抗体水平.统计学分析显示,测定的豚鼠PD50和牛PD50之间具有极...     

Cedivac-FMD can be used according to a marker vaccine principle

In this study, we investigated whether Cedivac-FMD, an emergency vaccine against foot-and-mouth disease (FMD), is suitable for use conjointly with a screening program intended to confirm freedom from disease in vaccinated herds based on evidence of virus replication in vaccinates. Different sets of sera were tested using the Ceditest FMDV-NS ELISA for the detection of antibodies against non-structural proteins (NSPs) of FMD virus. During a vaccine safety study, serum samples were collected from 10 calves, 10 lambs and 10 piglets following administration of a double dose and a repeat dose of high payload trivalent Cedivac-FMD vaccine. All serum samples collected both 2 weeks following the administration of a double dose as well as those collected 2 weeks after the single dose booster (given 2 weeks after the double dose) were negative in the Ceditest FMDV-NS ELISA. In a series of vaccine potency experiments, serum samples were collected from 70 vaccinated cattle prior to and following exposure to infectious, homologous FMD virus. When testing cattle sera collected 4 weeks after vaccination with a regular dose of monovalent >6 PD(50) vaccines, 1 of 70 animals tested positive in the NSP antibody ELISA. After infection with FMD virus, antibodies to NSP were detected in 59 of 70 vaccinated cattle and 27 of 28 non-vaccinated control animals within 7 days. Cedivac-FMD vaccines do not induce NSP antibodies in cattle, pigs or sheep following administration of a double dose or a repeat dose. FMD-exposed animals can be detected in a vaccinated group within 7-14 days. Because Cedivac-FMD does not induce NSP antibodies, the principle of 'marker vaccine' applies.     

A two year BTV-8 vaccination follow up: molecular diagnostics and assessment of humoral and cellular immune reactions

The compulsory vaccination campaign against Bluetongue virus serotype eight (BTV-8) in Germany was exercised in the state of Bavaria using three commercial monovalent inactivated vaccines given provisional marketing authorisation for emergency use. In eleven Bavarian farms representing a cross sectional area of the state the immune reactions of sheep and cattle were followed over a two year period (2008-2009) using cELISA, a serum neutralisation test (SNT) and interferon gamma (IFN-γ) ELISPOT. For molecular diagnostics of BTV genome presence two recommended real time quantitative RT-PCR protocols were applied. The recommended vaccination scheme led to low or even undetectable antibody titers (ELISA) in serum samples of both cattle and sheep. A fourfold increase of the vaccine dose in cattle, however, induced higher ELISA titers and virus neutralising antibodies. Accordingly, repeated vaccination in sheep caused an increase in ELISA-antibody titers. BTV-8 neutralising antibodies occurred in most animals only after multiple vaccinations in the second year of the campaign. The secretion of interferon gamma (IFN-γ) in ELISPOT after in vitro re-stimulation of PBMC of BTV-8 vaccinated animals with BTV was evaluated in the field for the first time. Sera of BTV-8 infected or vaccinated animals neutralising BTV-8 could also neutralise an Italian BTV serotype 1 cell culture adapted strain and PBMC of such animals secreted IFN-γ when stimulated with BTV-1.     

Relationship between the anti-FMD virus antibody reaction as measured by different assays, and protection in vivo against challenge infection.

The antibody response of cattle after vaccination against foot-and-mouth disease (FMD) virus was monitored using the serum neutralization test (SNT), the sandwich ELISA, liquid-phase ELISA, sandwich competition ELISA, liquid-phase competition ELISA, and the liquid-phase sandwich competition (blocking) ELISA. The competition ELISAs (in particular the "blocking" ELISA) were the most effective at detecting reactivity in these cattle sera. However, 95% of negative sera also competed in the most sensitive ELISA (the "blocking" ELISA) to titres of 1:32 (4% of the sera competed to a titre of 1:128). Comparisons between the different ELISAs, and between these ELISAs and the SNT, demonstrated that the tests were not measuring exactly the same reaction of antibody with FMD virus. With respect to the capacity of animals to resist FMD virus challenge, neither the SNT nor the competition ELISAs were consistently able to identify such animals. The anti-FMD virus antibody titres obtained could be classified into three zones; the "white zone" wherein antibody titres were high and donor animals likely to be protected; the "black zone" wherein antibody titres were low and donor animals likely to be susceptible to infection; the "grey zone" wherein the antibody titres were intermediary and no interpretation could be made with respect to protection. Assays such as ELISA and SNT cannot and do not measure immunological protection; they are a measure of antibody responses and nothing more, and should be interpreted in terms of the "three zone" phenomenon.     

Detection of carriers of foot-and-mouth disease virus among vaccinated cattle

To investigate and optimise detection of carriers, we vaccinated 15 calves with an inactivated vaccine based on foot-and-mouth disease virus (FMDV) A Turkey strain and challenged them and two further non-vaccinated calves with the homologous virus four weeks later. To determine transmission to a sensitive animal, we put a sentinel calf among the infected cattle from 60 days post-infection until the end of the experiment at 609 days post-infection. Samples were tested for the presence of FMDV, viral genome, specific IgA antibodies, antibodies against FMDV non-structural (NS) proteins or neutralising antibodies. Virus and viral genome was intermittently isolated from probang samples and the number of isolations decreased over time. During the first 100 days significantly more samples were positive by RT-PCR than by virus isolation (VI), whereas, late after infection more samples were positive by virus isolation. All the inoculated cattle developed high titres of neutralising antibodies that remained high during the entire experiment. An IgA antibody response was intermittently detected in the oropharyngeal fluid of 14 of the 17 calves, while all of them developed detectable levels of antibodies to NS proteins of FMDV in serum, which declined slowly beyond 34 days post-infection. Nevertheless, at 609 days after inoculation, 10 cattle (60%) were still positive by NS ELISA. Of the 17 cattle in our experiment, 16 became carriers. Despite frequent reallocation between a different pair of infected cattle no transmission to the sentinel calf occurred. It remained negative in all assays during the entire experiment. The results of this experiment show that the NS ELISA is currently the most sensitive method to detect carriers in a vaccinated cattle population.     

Protective effect of vaccination against experimental adenoviral pneumoenteritis in fattening lambs

The protective value of monovalent and bivalent adenovirus vaccines against pneumoenteritis was studied in fattening lambs. The monovalent vaccine was prepared from strain ORT/111 which is a sheep isolate, related to bovine adenovirus type 2. The bivalent vaccine contained virus strains ORT/111 and GY/14, which is a local isolate of ovine adenovirus type 1. After administration of monovalent vaccine higher titres of neutralizing antibodies (1:128–1:512) were found than after the bivalent vaccine (1:16–1:128).Following challenge with a homologous virus strain, the lambs vaccinated with monovalent vaccine remained healthy and did not shed the virus. Those vaccinated with the bivalent vaccine and challenged with GY/14 strain also remained healthy, but two out of five lambs shed virus in the faeces. Non-vaccinated challenged controls developed characteristic signs of the disease and shed the virus both in the nasal discharge and the faeces. From the results it can be concluded that experimental adenoviral pneumoenteritis of fattening lambs can be prevented by active immunization.     

Bovine respiratory syncytial virus-specific immune responses in cattle following immunization with modified-live and inactivated vaccines. Analysis of the specificity and activity of serum antibodies.

Cattle were immunized with vaccines containing modified-live or inactivated bovine respiratory syncytial virus (BRSV) and serum antibody responses were analyzed. Compared with preinculation values, at Day 14 after two biweekly immunizations with modified-live or inactivated vaccines there were significant increases in BRSV-specific titers in the sera of cattle that received both types of vaccines, as determined by a whole cell ELISA. Using a blocking ELISA and radioimmune precipitation it was determined that there was recognition of the fusion (F) protein by antibodies from cattle that received both types of BRSV antigens: however, virus neutralization assays revealed that only cattle that received modified live virus, either in monovalent or polyvalent vaccines, developed neutralizing antibodies to BRSV after two immunizations. These results indicate that inactivation of BRSV can lead to a dissociation between serological recognition of the F protein and virus neutralization in vaccinated cattle.     

Duration of immunity induced by an adjuvanted and inactivated equine influenza, tetanus and equine herpesvirus 1 and 4 combination vaccine

An adjuvanted vaccine containing inactivated equine influenza, herpesvirus antigens, and tetanus toxoid was administered to young seronegative foals of 8 months of age by deep intramuscular injection in the neck (Group A). The first two vaccinations were given 4 weeks apart. The third was administered 6 months later. Another group of foals (Group B) was vaccinated according to the same scheme at the same time with monovalent equine herpes virus (EHV) vaccine (EHV1.4) vaccine. Antibody responses to the equine influenza (single radial haemolysis; SRH) and tetanus (ToBi ELISA) components of the vaccines were examined from first vaccination until 1 year after the third vaccination. The influenza components of the combination vaccine induced high antibody titres at two weeks after the second vaccination whereafter titres declined until the time of the third vaccination. After the third vaccination, the titres rose rapidly again to remain high for at least 1 year. Antibody titres against tetanus peaked only after the third vaccination but remained high enough to offer protective immunity for at least 1 year. Foals vaccinated with monovalent EHV1.4 remained seronegative for influenza and tetanus throughout the study. Four and a half months after the third vaccination of groups A and B, a third group of animals was vaccinated twice with monovalent EHV1.4 vaccine 4 weeks apart (Group C). Two weeks after the administration of the second dose in the later group, all groups (A, B, C and an unvaccinated control group D) were challenged with EHV-4. Vaccinated foals (Group A, B, C) showed a clear reduction of clinical symptoms and virus excretion after EHV-4 challenge compared with the unvaccinated control foals. No difference could be demonstrated among the vaccinated groups, suggesting that the combination vaccine protects as well as the monovalent vaccine. In EHV1.4-vaccinated foals both antigenic fractions induced clear protection up to 6 months after vaccination (9). It can therefore be anticipated that the efficacy of the combination vaccine against EHV-1 challenge is similar to the efficacy against EHV-1 induced by EHV1.4 vaccination.     

Presence of antibodies to non-structural proteins of foot-and-mouth disease virus in repeatedly vaccinated cattle

For the purpose of removing infected animals by detecting humoral immune responses to non-structural proteins of the foot-and-mouth disease (FMD) virus, antibodies induced by contaminated residual non-structural proteins contained in less pure FMD vaccine can be problematic for serological screening. The aim of the present study was to measure the possible presence of antibodies against these non-structural proteins in repeatedly vaccinated calves and beef cattle. Five imported FMD vaccines were examined using two commercial ELISA kits, UBI FMDV NS EIA and Ceditest FMDV-NS, for serological testing. After five doses of vaccination, the serum of one calf tested positive, and two vaccines induced a significant increase in anti-3ABC antibodies in calves. This finding demonstrated that a positive reaction to non-structural proteins due to impurities in the FMD vaccine was detectable using commercial tests. A low percentage of field sera sampled from beef cattle in Kinmen also tested positive, but the key factor resulting in the positive reactions could not be positively identified based on our data.     

Seasonal variations in the formation of virus-neutralizing antibodies in cattle after vaccination against foot-and-mouth disease

The GDR in 1977 had been the first country to introduce a laboratory method for potency testing of FMD vaccines on the basis of secured correlations between titres of virus-neutralising serum antibodies of immunised cattle, on the one hand, and their probit-transformed protection against FMD, on the other. This is now the only state-registered method for potency testing. The method has ever since worked well all over the place. An evaluation of results obtained between 1982 and 1988 revealed seasonal variations of anti-FMD immunogenesis in cattle. FMD immunisations from February to July proved more effective than those performed in the rest of the year, in that higher antibody titres were built up within 14 days from vaccination. These results were confirmed by rates of protection of cattle recorded from direct potency testing of immunised animals, between 1969 and 1977.     

Safety, efficacy and cross-protectivity of a live intranasal aerosol haemorrhagic septicaemia vaccine

The safety, efficacy and cross-protectivity of a live intranasal aerosol haemorrhagic septicaemia vaccine containing Pasteurella multocida serotype B:3,4 were tested in young cattle and buffaloes in Myanmar, where more than 1.5 million animals had been inoculated with this vaccine between 1989 and 1999. A recommended dose of 2 x 10(7) viable organisms was used for the efficacy test. The administration of 100 times the recommended dose to 50 cattle and 39 buffalo calves was innocuous. Seven months after they were vaccinated, three of three buffaloes were protected and 12 months after they were vaccinated, three of four buffaloes were protected against a subcutaneous challenge with serotype B:2 which killed three of three unvaccinated buffaloes. Twelve months after they were vaccinated, eight of eight cattle survived a serotype B:2 challenge, which killed four of four unvaccinated controls. The vaccinated cattle had developed serum antibodies detectable by the passive mouse protection test. Indirect haemagglutination tests on sera taken from cattle 10 days and five weeks after they were vaccinated showed high titres of antibodies. The serum of vaccinated cattle cross-protected passively immunised mice against infection with P. multocida serotypes E:2, F:3,4 and A:3,4.     

Testing the effectiveness of foot-and-mouth disease vaccines: the relationship between test infection results and corresponding neutralization titers of vaccinated cattle

In the course of vaccine controls, the potency of 25 foot-and-mouth disease (FMD) vaccines was tested quantitatively in parallel in cattle using the intradermal infection and the determination of the SN titres. More than 95% of the vaccinated cattle with SN titres of greater than 1:20 were protected from generalized FMD, regardless of the virus type tested. 61.5% of the vaccinated cattle with SN titres less than or equal to 1:20 were not protected and developed generalized FMD. Comparison of the PD50 values calculated from the results of the intradermal infection and the corresponding SN titres (minimum protection titre greater than 1:20) showed that the results were in complete agreement in 56% of the tested vaccines. In a further 32% of vaccines, the PD50 calculated from the SN titre was slightly below that for the intradermal infection, in the remaining 12% it was somewhat above. The possibility of using the minimum titre determination for testing a vaccine and the significance of this titre as an expression of protection by vaccination are discussed.     

Evaluation of an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of infectious bovine rhinotracheitis infection, with results of a preliminary survey

An indirect enzyme linked immunosorbent assay (ELISA) procedure was evaluated against the serum neutralisation test (SNT) for the detection of antibodies to infectious bovine rhinotracheitis virus (bovine herpesvirus type l), using 2028 sera from 166 dairy and 172 beef cattle herds. The results showed the ELISA to give high levels of agreement with the SNT in classifying positive and negative sera (98% and 97% respectively). Such disagreements as did occur involved weakly reactive sera with SNT titres of % or less. A number of sera (n=123) with trace neutralising activity of doubtful diagnostic significance were found to give marginal reactivity with ELISA. ELISA absorbance values were found to be highly correlated with SNT titres (r=0.909) on an overall basis, though agreements were lower with individual sera. The ELISA procedure was quicker, cheaper, and detected more reactors than the SNT. It also allowed results to be obtained with a number of sera which were unsuitable for testing by SNT because of their cytotoxic nature. Analysis of ELISA results showed reactors to be present in 57% of tested sera, representing 81% of cattle herds. Reactor rates for sera and herds in the South Island, (37% and 58%), were significantly lower than for those in the North Island (64% and 88%). Antibody prevalence was also found to be significantly lower in districts having a low annual rainfall (<850 mm), and to be lower in beef cattle than in dairy cattle. A surprising exception to the latter occurred in low rainfall districts, where dairy cattle showed significantly lower reactor rates than local beef animals.     

Response to and efficacy of vaccination against eastern equine encephalomyelitis virus in emus

OBJECTIVE: To evaluate humoral immune responses of emus vaccinated with commercially available equine polyvalent or experimental monovalent eastern equine encephalomyelitis (EEE) virus and western equine encephalomyelitis (WEE) virus vaccines and to determine whether vaccinated emus were protected against challenge with EEE virus. DESIGN: Cohort study. ANIMALS: 25 emus. PROCEDURE: Birds were randomly assigned to groups (n = 5/group) and vaccinated with 1 of 2 commercially available polyvalent equine vaccines, a monovalent EEE virus vaccine, or a monovalent WEE virus vaccine or were not vaccinated. Neutralizing antibody responses against EEE and WEE viruses were examined at regular intervals for up to 9 months. All emus vaccinated with the equine vaccines and 2 unvaccinated control birds were challenged with EEE virus. An additional unvaccinated bird was housed with the control birds to assess the possibility of contact transmission. RESULTS: All 4 vaccines induced detectable neutralizing antibody titers, and all birds vaccinated with the equine vaccines were fully protected against an otherwise lethal dose of EEE virus. Unvaccinated challenged birds developed viremia (> 10(9) plaque-forming units/ml of blood) and shed virus in feces, oral secretions, and regurgitated material. The unvaccinated pen-mate became infected in the absence of mosquito vectors, presumably as a result of direct virus transmission between birds. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that emus infected with EEE virus develop a high-titer viremia and suggest that they may serve as important virus reservoirs. Infected emus shed EEE virus in secretions and excretions, making them a direct hazard to pen-mates and attending humans. Commercially available polyvalent equine vaccines protect emus against EEE virus infection.